Antibodies Against IL-1 Beta

ABSTRACT

The present invention relates to anti-IL-1 beta antibodies and in particular to monovalent high potency IL-1 beta-binding antibody fragments being highly stable. Such antibodies can be used in the treatment of inflammatory and other diseases as well as in diagnostics. Also provided are related nucleic acids, vectors, cells, and compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/896,309, filed on Feb. 14, 2018, which is a divisional of U.S.application Ser. No. 14/740,434, filed on Jun. 16, 2015 (now U.S. Pat.No. 9,914,772), which is a continuation of International Application No.PCT/EP2013/076831, which designated the United States and was filed onDec. 17, 2013, published in English, which claims the benefit of U.S.Provisional Application No. 61/738,223, filed on Dec. 17, 2012. Thisapplication claims priority under 35 U.S.C. § 119 or 365 to EPApplication No. 13000746.1, filed Feb. 14, 2013. The entire teachings ofthe above applications are incorporated herein by reference.

FIELD OF INVENTION

The invention relates to humanized anti-IL-1 beta antibodies, inparticular monovalent, highly potent and stable anti-IL-1 beta antibodyfragments applicable for therapeutic uses. The invention also relates tonucleic acids encoding such antibodies and antibody fragments, vectors,host containing such sequences, pharmaceutical and diagnosticcompositions comprising the antibodies and antibody fragments or nucleicacids, and uses thereof.

BACKGROUND OF THE INVENTION

Interleukin 1 beta (IL-1 beta) is a pro-inflammatory cytokine which isexpressed as a precursor called pro-IL-1 beta by activated macrophages,monocytes and dendritic cells. The precursor is cleaved by caspase-1 toform the biologically active and secreted form of IL-1 beta. Binding ofIL-1 beta binds to its receptor, IL-1 receptor type I (IL-1R1), allowsthe recruitment of a second receptor subunit, the IL-1R accessoryprotein (IL-1RAP). The formed complex is competent of signaltransduction. Being a key mediator in the inflammatory response, thecytokine affects a number of cellular activities such as cellproliferation, differentiation, and apoptosis. Therefore, IL-1 beta isconsidered an important target for a variety of drugs.

Several antibodies to IL-1 beta have previously been reported. R&DSystems, Inc. produces and sells the murine anti-human IL-1 beta/IL-1F2antibody MAB201 (R&D Systems, Inc., cat. no. MAB201), a full-lengthimmunoglobulin IgG1, which is produced in hybridoma culture.

MAB201, which displays a half-maximum neutralizing potency range from6.6 to 20 pM according to the supplier, has been humanized by graftingits CDRs onto human kappa chain germline sequence and gamma chain VH-2acceptor sequences (US20030026806, AMGEN, Inc.) to yield a full-lengthimmunoglobulin.

XOMA052, also known as AB7 or gevokizumab, is a humanized IgG₂, i.e, afull-length immunoglobulin (OWYANG, A., et al. XOMA 052, a potent,high-affinity monoclonal antibody for the treatment of IL-1beta-mediated diseases. mAbs 2011, vol. 3, p. 49-60; EP 1899378 B, XOMATECHNOLOGY, Ltd.). Its variable domains were humanized to match a humankappa 1 light chain and a human VH2 heavy chain. Its CDRs are identicalto MAB201 with the exception of one conservative point mutation inCDR-H2.

Typically, after CDR-grafting for, e.g., humanization purposes, anantibody having accommodated new CDRs from a CDR-donor antibodyexperiences a drop in potency when compared to the CDR-donor antibody.It is therefore a true challenge, if-successful at all, to furtherengineer the generated CDR-acceptor antibody such that the potency isclose or equal to the CDR-donor antibody.

Moreover, for use in medical treatments it is mandatory, besidesproviding a good biological potency of the antibody, to generateantibodies with enhanced drug-like properties, e.g., high stability andsufficient solubility.

Hence, there is a need for novel antibodies and in particular antibodyfragments, which overcome the disadvantages of the existing antibodiesin the art. The invention provides such compounds, as well as methodsfor preparing and using these.

SUMMARY OF THE INVENTION

In a first aspect, a humanized antibody against IL-1 beta is provided.

In one embodiment, said humanized antibody comprises a variable lightchain framework sequence having at least 90% identity to SEQ ID No: 8;and a variable heavy chain framework sequence having at least 90%identity to SEQ ID No: 12.

Additionally or alternatively, said antibody comprises variable lightchain CDR-L1, CDR-L2 and CDR-L3 sequences as set forth in SEQ ID Nos: 1,2, and 3, respectively, or variants thereof, and wherein

-   -   position 1 of the light chain is an aspartic acid residue (D);    -   position 3 of the light chain is a glutamine residue (Q);    -   position 20 of the light chain is a threonine residue (T);    -   position 99 of the light chain is a glutamic acid residue (E);    -   position 105 of the light chain is a phenylalanine residue (F);    -   position 146 of the light chain is a glutamic residue (E);    -   position 147 of the light chain is an isoleucine residue (I);    -   position 148 of the light chain is a lysine residue (K); and/or    -   position 149 of the light chain is a arginine residue (R),        according to AHo numbering.

Additionally or alternatively, said antibody comprises CDR-H1, CDR-H2and CDR-H3 sequences set forth in SEQ ID Nos: 4, 21, and 6,respectively, or variants thereof, and wherein

-   -   position 24 of the heavy chain is an alanine residue (A);    -   position 25 of the heavy chain is a phenylalanine residue (F);    -   position 44 of the heavy chain is an isoleucine residue (I);    -   position 56 of the heavy chain is an serine residue (S);    -   position 82 of the heavy chain is a lysine residue (K);    -   position 86 of the heavy chain is an arginine residue (R);        and/or    -   position 105 of the heavy chain is a phenylalanine residue (F),        according to AHo numbering.

Preferably, said antibody fragment has a VH domain of human subtype VH3or VH1b.

4) The antibodies provided herein are highly stable, i.e., they remainmonomeric for prolonged periods of time. This applies in particular tothe antibody fragments and more particularly to the scFvs disclosedherein. Stability parameters are crucial factors for providing a viabledrug. The more stable a drug, the longer the shelf half-life time.Unstable antibodies tend to dimerize or oligomerize and evenprecipitate, thereby decreasing shelf-life and finally becoming lesssuitable for pharmaceutical applications because of, e.g., increasedimmunogenicity.

For certain therapeutic indications, small antibody fragments haveadvantages over full-length immunoglobulins because of their smallersize and the lack of the constant region Fc of immunoglobulins. Forexample, scFv are capable of more efficiently penetrating tissues due totheir small size. Further, they display a decreased retention in thesystemic circulation as they are unable to bind to Fc receptors such asFcRn eventually leading to higher renal clearance rates. Thesecharacteristics of good tissue penetration with subsequent evendistribution in the tissue and the rapid elimination of small antibodyfragments such as scFv from the systemic circulation are particularlyadvantageous for both chronic local/topical as well as acute systemicdiseases. This practical utility has however been severely limited inthe past by low stability and low biological potency of recombinanthumanized scFv.

The antibodies provided herein exert very high inhibitory potencies withregard to human IL-1 beta. Monovalent antibody fragments having potencyvalues in the pM-range are particular and not routinely obtained. Inaddition and typically, an antibody loses affinity to its target uponhumanization when compared to the parent non-human antibody. It istherefore a challenge to humanize an antibody such that the affinityparameters are close or equal to the parent antibody. This isparticularly true for monovalent antibody fragments which comprise onlyone variable light and heavy chain, and therefore bind to the targetless strongly than bivalent antibodies displaying two light and heavychains.

A biologically very potent antibody is particularly useful since itallows, e.g., the administration of low amounts of drug to the patient,thereby decreasing the overall costs of treatment. In addition, a morecomplete neutralization of the molecular target of the disease isrendered feasible.

Moreover, different, novel application routes in animal models as wellas in human therapy can be envisioned when applying highest potencyantibodies. For example, as to topically applied drugs, although thedelivery efficacy may be limited due to the barrier function of the skinstratum corneum and/or other biological structures, the efficacy oftreatment is restored by the high potency of the otherwise limitedquantity of drug molecules that passes such physiological barriers.

Often, the high amount of a less potent drug which needs to beadministered to achieve similar pharmacodynamic effects as with a morepotent drug, translates into much higher intravenous or subcutaneousapplication volumes. Such higher application volumes are a disadvantagefor use in animals and humans for two reasons: firstly, theimpracticality of treating patients with a high volume of drug, andsecondly, antibodies are expensive per unit of mass.

Lower quantities of antibodies required for treatment translate intolower production costs for the drug. In particular antibody fragmentsare amenable to low production costs since the use of, e.g., bacterialor yeast culture systems is leading to lower costs than with mammalianexpression systems typically used for the production of full-lengthimmunoglobulins. The combination of smaller quantities of drug to beadministered and cheaper manufacturing processes opens the possibilityof more cost-efficient medicines per patient. Thus, a larger number ofpatients may benefit from such drug.

Stability parameters are other factors crucial for providing a viablemedicament. The more stable an antibody drug, the longer the shelfhalf-life time. The antibodies provided herein are highly stable, i.e.,they remain monomeric for prolonged periods of time and also at highconcentrations, having the advantage of smaller volumes ofadministration.

In another aspect; a nucleic acid molecule encoding the antibodiesabove, a vector comprising said nucleic acid molecule and/or host cellscomprising said nucleic acid molecule or said vector are provided.

Also provided is a composition comprising the antibody above, thenucleic acid above, the vector above or the host cell above; and furthera suitable carrier, diluent or excipient.

Further provided is a method of treating an IL-1 beta-mediated diseasecomprising administering to a subject in need thereof saidpharmaceutical composition.

The antibodies described herein can, e.g., be used

(i) as medicament, e.g., in the treatment of an IL-1 beta-mediateddisease;

(ii) in diagnostics;

(iii) in cosmetics; and/or

(iv) for detection purposes.

Further provided is a method of producing the antibodies disclosedherein comprising the steps of

(i) cultivating the host cell above so that the antibody is expressed;

(ii) recovering the antibody; and

(iii) optionally purifying the antibody.

Optionally, said method may further comprise at least one step ofchemical synthesis.

Further provided is a method of producing the antibodies disclosedherein comprising the steps of

-   -   (a) providing a cell-free system,    -   (b) providing a nucleic acid product template,    -   (c) allowing for transcription and translation of said nucleic        acid product template, thereby expressing the antibody;    -   (d) recovering the antibody; and    -   (e) optionally purifying the antibody.        Said method may optionally comprise at least one step of        chemical synthesis.

Also provided is an in vivo or an in vitro method of detecting thepresence of IL-1 beta in a biological sample comprising the steps of

(i) contacting said biological sample with an antibody disclosed hereinunder conditions permissive for binding to IL-1 beta, and

(ii) detecting whether a complex is formed with IL-1 beta.

Further provided is a kit comprising the antibody above together with apackaged combination of reagents with instructions.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A depicts alignments of light chain sequences (VL) of anti-IL-1beta scFv DLX2260, DLX2289, DLX2332, DLX2295 and DLX2296 correspondingto SEQ ID Nos, 17, 16, 20, 19 and 18, respectively. FIG. 1B showsalignments of the corresponding heavy chain sequences (VH). Amino acidsdiffering at the same position are marked bold, CDR sequences areunderlined.

FIG. 2A shows the results of an ELISA to determine the binding ofDLX2260 and DLX2289 to biotinylated rhIL-1 beta at variousconcentrations. Absorbance differences at a wavelength of 450 nm areplotted as function of the concentration of biotinylated IL-1 beta givenin ng/ml. Squares: DLX2260; circles; DLX2289.

FIG. 2B shows the inhibition of IL-1 beta induced proliferation ofD10.G4.1 cells by three different anti-IL-1 beta antibodies. Absorbancedifferences at a wavelength of 450 nm are plotted as function of theconcentration of scFv given in ng/ml. Squares: DLX2260; circles:DLX2289; triangles: MAB201.

FIG. 3A shows the binding of DLX2295 and DLX2296 to rhIL-1 beta in anELISA. Absorbance differences at a wavelength of 450 nm are plotted as afunction of the concentration of scFv given in ng/ml. Squares: controlscFv; circles: DLX2296; triangles: DLX2295. The result for the sameexperiment performed with DLX2332 is given in FIG. 3B. Absorbancedifferences at a wavelength of 450 nm are plotted as a function of theconcentration of scFv given in ng/ml. Circles: control scFv; triangles:DLX2332.

FIG. 4 shows the inhibition of rhIL-1 beta biological activity in ahuman fibroblast assay. The potency of scFvs DLX2260 and DLX2296 as wellas of the control antibody MAB201 is compared. The y-axis indicates theamount of released IL-6 from human fibroblasts in pg/ml; the x-axisindicates the concentration of applied antibodies in pM. Squares:MAB201; circles: DLX2260; triangles: DLX2296.

FIG. 5 shows the inhibition of rhIL-1 beta biological activity byDLX2295 in a human fibroblast assay. In the same experiment, thepositive control antibodies MAB201 and Ilaris® were analyzed. The y-axisindicates the amount of released IL-6 from human fibroblasts in pg/ml;the x-axis indicates the concentration of applied antibodies in pM.Squares: MAB201; triangles: DLX2295; circles: Ilaris®.

FIG. 6 shows the IL-1 beta neutralization capacity of DLX2332 incomparison to DLX2295 in a human fibroblast assay. The y-axis indicatesthe amount of released IL-6 from human fibroblasts in pg/ml; the x-axisindicates the concentration of applied antibodies in pM. Squares:DLX2295; circles: DLX2332.

FIG. 7 shows the definition of the CDR-H1 region as used herein and inthe Kabat numbering scheme. Arrows indicate the CDR-H1 residuesaccording to the Kabat definition (above) or as used herein (below).

5. DETAILED DESCRIPTION

So that the invention may be more readily understood, certain terms willbe first defined. Unless otherwise defined within the specification, alltechnical and scientific terms used herein have their art-recognizedmeaning. Although similar or equivalent methods and materials to thosedescribed herein can be used in the practice or testing of theinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict the present specification including definitions willprevail. The materials, methods, and examples are illustrative only andnot intended to be limiting.

Within the scope of the present invention the term “antibody” refers tofull-length immunoglobulins as well as to fragments thereof. Suchfull-length immunoglobulins may be monoclonal, polyclonal, chimeric,humanized, veneered or human antibodies.

Such antibody can be monovalent or multivalent, i.e. having one or moreantigen binding sites. Non-limiting examples of monovalent antibodiesinclude scFv, Fab fragments, dAb, VHH and nanobodies. A multivalentantibody can have two, three, four or more antigen binding sites wherebyone or more different antigens can be recognized. Full-lengthimmunoglobulins, F(ab′)₂ fragments, bis-scFv and diabodies arenon-limiting examples of multivalent antibodies; in said exemplarymultivalent antibodies, two binding sites are present, i.e. the antibodyis bivalent.

In one embodiment the multivalent antibody is bispecific, i.e. theantibody is directed against two different targets or two differenttarget sites on one target molecule. Bispecific antibodies are, e.g.,reviewed in MÜLLER, D. and Kontermann, R. E. Bispecific antibodies.Edited by DÜBEL, S. Weinheim: Wiley-VCH, 2007. ISBN 3527314539. p.345-378. In another embodiment the multivalent antibody comprises morethan two, e.g., three or four different binding sites for three or four,respectively, different antigens. Such antibody is multivalent andmultispecific, in particular tri- or tetra-specific, respectively.

“Antibody fragments” comprise portions of a full-length immunoglobulinessentially retaining the antigen-specificity of said immunoglobulin.Many but not all antibody fragments lack at least partially the constantregion (Fc region) of the full-length immunoglobulin. In someembodiments antibody fragments are produced by proteolytic digestion ofthe full-length immunoglobulin. An antibody fragment may also be asynthetic or recombinant construct comprising parts of theimmunoglobulin or immunoglobulin chains (see e.g. HOLLIGER, P. andHudson, J. Engineered antibody fragments and the rise of single domains.Nature Biotechnology 2005, vol. 23, no. 9, p. 1126-1136). Examples ofantibody fragments, without being limited to, include scFv, Fab, Fv,Fab′, F(ab′)₂ fragments, dAb, VHH, nanobodies, V(NAR) or minimalrecognition units.

“Single chain variable fragments” or “single chain antibodies” or “scFv”are one type of antibody fragments. scFv are fusion proteins comprisingthe VH and VL of immunoglobulins connected by a linker. They thus lackthe constant Fc region present in full-length immunoglobulins, butretain the antigen-specificity of the original immunoglobulin.

The “IC₅₀” or “half-maximum inhibitory concentration” is a measure ofantagonist drug potency and describes quantitatively the effectivenessof a compound to inhibit a biological or biochemical function. Thismeasure indicates how much of such compound is needed to inhibit by 50%a certain biological or biochemical process. If residual activity isobserved, the IC₅₀ value is the concentration of the compound at theinflection point of the measured inhibition curve. Although no directindicator of affinity, both values are correlated via the Cheng-Prusoffequation (CHENG Y. and Prusoff W. H. Relationship between the inhibitionconstant (Ki) and the concentration of inhibitor which causes 50 percentinhibition (150) of an enzymatic reaction. Biochemical Pharmacology1973, vol. 22, p. 3099-3108; RAMMES, G., et al. Identification of adomain which affects kinetics and antagonistic potency of clozapine at5-HT3 receptors. PLOS one 2009, vol. 4, p. 1-14; ZHEN, J., et al.Concentration of receptor and ligand revisited in a modified receptorbinding protocol for high-affinity radioligands: [³H] spiperorie bindingto D2 and D3 dopamine receptors. Journal of Neuroscience Methods 2010,vol. 188, p. 32-38).

The term “IL-1 beta specific binding” as used herein describes that anantibody binds to IL-1 beta with higher affinity than to a structurallydifferent antigen which does not comprise the IL-1 beta epitope to whichsuch anti-IL-1 beta antibody binds. Specific binding is reflected by adissociation equilibrium constant (K_(D)) of lower than 1 micromolar.This constant can be determined, e.g., using Quartz Crystal Microbalance(QCM) in an Attana instrument, or Surface Plasmon Resonance (SPR)technology in a BIACORE instrument.

As used herein, “IL-1 beta” refers to the molecule as described in e.g.,Dinarello C. A., A clinical perspective of IL-1 beta as the gatekeeperof inflammation. Eur. J. Immunol. 2011, vol. 41, p. 1203-1217. “hIL-1beta” as used herein refers to human IL-1 beta. “rIL-1 beta” refers torecombinant IL-1 beta. Recombinant IL-1 beta may or may not have anamino terminal methionine residue, depending upon the method by which itis prepared. “rhIL-1” beta refers to recombinant human IL-1 beta, rhIL-1beta may e.g. be obtained from Peprotech, USA, cat. no. 200-01B. IL-1beta may also be obtained by isolation from biological samples of humanor non-human origin.

“Humanized” antibody refers to a full-length immunoglobulin or afragment thereof comprising one or more, typically all six CDR regionsof a non-human parent antibody or variant thereof, and in which theframework may, e.g., be (i) a human framework, potentially comprisingone or more framework residues of the non-human parent antibody, or (ii)a framework from a non-human antibody modified to increase similarity tonaturally produced human frameworks. Methods of humanizing antibodiesare known in the art, see, e.g., LEGER, O. and Saldanha, J. AntibodyDrug Discovery. Edited by WOOD, C. London: Imperial College Press, 2011.ISBN 1848166281, p. 1-23, whereas the functionality is stillunpredictable.

“Framework” (FR) refers to the scaffold of the variable antibody domain,either the variable light chain (VL) or variable heavy chain (VH), whichembed the respective CDRs. A VL and/or VH framework typically comprisesfour framework sections, FR1, FR2, FR3 and FR4, flanking the CDRregions. Thus, as known in the art, a VL has the general structure:(FR-L1)-(CDR-L1)-(FR-L2)-(CDR-L2)-(FR-L3)-(CDR-L3)-(FR-L4), whereas a VHhas the general structure:(FR-H1)-(CDR-H1)-(FR-H2)-(CDR-H2)-(FR-H3)-(CDR-H3)-(FR-H4).

“Complementarity determining region” (CDR) refers to the hypervariableregions of the antibody which mainly contribute to antigen binding.Typically, an antigen binding site comprises six CDRs embedded into aframework. Herein, the CDRs of the VL are referred to as CDR-L1, CDR-L2and CDR-L3 whereas the CDRs of the VH are referred to as CDR-H1, CDR-H2and CDR-H3. These can be identified as described in KABAT, E. A., et al.Sequences of Proteins of Immunological Interest. 5th edition. Edited byU.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991. p.91-3242. CDR-H1 as used herein, however, differs from the Kabatdefinition in that it starts with position 27 and ends prior to position36 (see FIG. 7 for illustration).

As used herein, the numbering system to identify amino acid residuepositions in the VH and VL of the antibody corresponds to the“AHo”-system described by HONEGGER, A. and Pluckthun, A. Yet anothernumbering scheme for immunoglobulin variable domains: An automaticmodelling and analysis tool. Journal of Molecular Biology 2001, vol.309, p. 657-670. The publication further provides conversion tablesbetween the AHo and the Kabat system (KABAT, E. A., et al. Sequences ofProteins of Immunological Interest. 5th edition. Edited by U.S.DEPARTMENT OF HEALTH AND HUMAN SERVICES. NIH Publications, 1991. p.91-3242).

An “isolated” antibody or nucleic acid is one being identified andseparated from at least one component of its natural environment.

The term “identity” as used herein refers to the sequence match betweentwo proteins or nucleic acids. The protein or nucleic acid sequences tobe compared are aligned to give maximum identity, for example usingbioinformatics tools such as EMBOSS Needle (pair wise alignment;available at www.ebi.ac.uk). When the same position in the sequences tobe compared is occupied by the same nucleobase or amino acid residue,then the respective molecules are Identical at that very position.Accordingly, the “percent identity” is a function of the number ofmatching positions divided by the number of positions compared andmultiplied by 100%. For instance, if 6 out of 10 sequence positions areidentical, then the identity Is 60%. The percent identity between twoprotein sequences can, e.g., be determined using the Needleman andWunsch algorithm (NEEDLEMAN, S. B. and Wunsch, C.D. A general methodapplicable to the search for similarities in the amino acid sequence oftwo proteins. Journal of Molecular Biology 1970, vol. 48, p. 443-453)which has been incorporated into EMBOSS Needle, using a BLOSUM62 matrix,a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “endgap penalty”, an “end gap open penalty” of 10 and an “end gap extendpenalty” of 0.5. The % identity or similarity is typically determinedover the entire length of the query sequence on which the analysis isperformed. Two molecules having the same primary amino acid or nucleicacid sequence are identical irrespective of any chemical and/orbiological modification. For example, two antibodies having the sameprimary amino acid sequence but different glycosylation patterns areidentical by this definition. In case of nucleic acids, for example, twomolecules having the same sequence but different linkage components suchas thiophosphate instead of phosphate are identical by this definition.

“Similar” protein sequences are those which, when aligned, share similaramino acid residues and most often, but not mandatorily, identical aminoacid residues at the same positions of the sequences to be compared.Similar amino acid residues are grouped by chemical characteristics ofthe side chains into families. Said families are described below for“conservative amino acid substitutions”. The “percent similarity”between sequences is the number of positions that contain identical orsimilar residues at the same sequence positions of the sequences to becompared divided by the total number of positions compared andmultiplied by 100%. For instance, if 6 out of 10 sequence positions haveidentical amino acid residues and 2 out of 10 positions contain similarresidues, then the sequences have 80% similarity. The similarity betweentwo sequences can, e.g., be determined using EMBOSS Needle.

A “variant” refers to an amino acid or nucleic acid sequence whichdiffers from the parental sequence by virtue of addition (includinginsertions), deletion and/or substitution of one or more amino acidresidues or nucleobases while retaining at least one desired activity ofthe parent sequence disclosed herein. In the case of antibodies suchdesired activity may include specific antigen binding. As to a variantnucleic acid sequence, the encoded antibody retains at least one desiredactivity of the parent antibody as described above. Variants may benaturally occurring, such as allelic or splice variants, or may beartificially constructed.

As used herein, the term “conservative modifications” refers tomodifications that are physically, biologically, chemically orfunctionally similar to the corresponding reference, e.g., similar size,shape, electric charge, chemical properties, including the ability toform covalent or hydrogen bonds, or the like. Such conservativemodifications include, but are not limited to, one or more nucleobaseand amino acid substitutions.

For example, conservative amino acid substitutions include those inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Amino acid residues being non-essentialwith regard to binding to an antigen can, e.g., be replaced with anotheramino acid residue from the same side chain family, e.g., serine may besubstituted for threonine. Amino acid residues are usually divided intofamilies based on common, similar side-chain properties, such as:

-   -   1. nonpolar side chains (e.g., glycine, alanine, valine,        leucine, isoleucine, methionine),    -   2. uncharged polar side chains (e.g., asparagine, glutamine,        serine, threonine, tyrosine, proline, cysteine, tryptophan),    -   3. basic side chains (e.g., lysine, arginine, histidine,        proline),    -   4. acidic side chains (e.g., aspartic acid, glutamic acid),    -   5. beta-branched side chains (e.g., threonine, valine,        isoleucine), and    -   6. aromatic side chains (e.g., tyrosine, phenylalanine,        tryptophan, histidine).        A conservative substitution may also involve the use of a        non-natural amino acid.

“Non-conservative substitutions”, i.e. exchanging members of one familyagainst members of another family, may lead to substantial changes,e.g., with respect to the charge, dipole moment, size, hydrophilicity,hydrophobicity or conformation of the antibody, which may lead to asignificant drop in the binding activity, in particular if amino acidsare affected that are essential for binding to the target molecule. Anon-conservative substitution may also involve the use of a non-naturalamino acid.

Conservative and non-conservative modifications can be introduced intoparental antibodies by a variety of standard techniques known in the artsuch as combinatorial chemistry, site-directed DNA mutagenesis,PCR-mediated and/or cassette mutagenesis, peptide/protein chemicalsynthesis, chemical reactions specifically modifying the parentalantibody. The variants can be tested by routine methods for theirchemical, biological, biophysical and/or biochemical properties.

Nucleic acid hybridization reactions can be performed under conditionsof different stringency. “Stringent conditions” are widely known andpublished in the art. Typically, during the hybridization reaction aSSC-based buffer can be used in which SSC is 0.15 M NaCl and 15 mMcitrate buffer having a pH of 7:0. Increasing buffer concentrations andthe presence of a denaturing agent increase the stringency of thehybridization step. For example, high stringency hybridizationconditions can involve the use of (i) 50% (vol/vol) formamide, 5×SSC(0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8),0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon spermDNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C. with washesat 42° C. in 0.2×SSC and 0.1% SDS; (ii) 50% (vol/vol) formamide with0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mMsodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mMsodium citrate at 42° C., or (iii) 10% dextran sulfate, 2×SSC, and 50%formamide at 55° C., followed by a high-stringency wash consisting of0.1×SSC containing EDTA at 55° C. Additionally or alternatively, one,two or more washing steps using wash solutions of low ionic strength andhigh temperature can be included in the hybridization protocol using,for example, 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodiumdodecyl sulfate at 50° C.

Various aspects of the invention are described in further detail in thefollowing subsections. It is understood that the various embodiments,preferences and ranges may be combined at will. Further, depending ofthe specific embodiment, selected definitions, embodiments or ranges maynot apply.

In a first aspect, the invention provides humanized antibodies bindingIL-1 beta.

In one embodiment, said humanized antibody comprises a variable lightchain sequence having at least 90% sequence identity to SEQ ID No: 8;and a variable heavy chain sequence having at least 90% sequenceidentity to SEQ ID No: 12.

Also provided is a humanized antibody binding IL-1 beta comprisingCDR-H1, CDR-H2 and CDR-H3 sequences set forth in SEQ ID Nos: 4, 21, and6, respectively, or variants thereof, and wherein

-   -   position 24 of the heavy chain is an alanine residue (A);    -   position 25 of the heavy chain is a phenylalanine residue (F);    -   position 44 of the heavy chain is an isoleucine residue (I);    -   position 56 of the heavy chain is an serine residue (S);    -   position 82 of the heavy chain is a lysine residue (K);    -   position 86 of the heavy chain is an arginine residue (R);        and/or    -   position 105 of the heavy chain is a phenylalanine residue (F),        according to AHo numbering.

Said antibody may further comprise

a serine residue (S) at position 12 of the heavy chain,

a serine residue (S) at position 103 of the heavy chain, and

a threonine residue (T) at position 144 of the heavy chain, according toAHo numbering.

Additionally or alternatively, the antibody comprises variable lightchain CDR-11, CDR-L2 and CDR-L3 sequences as set forth in SEQ ID Nos: 1,2; and 3, respectively, or variants thereof, and wherein

-   -   position 1 of the light chain is an aspartic acid residue (D);    -   position 3 of the light chain is a glutamine residue (Q);    -   position 20 of the light chain is a threonine residue (T);    -   position 99 of the light chain is a glutamic acid residue (E);    -   position 105 of the light chain is a phenylalanine residue (F);    -   position 146 of the light chain is a glutamic residue (E);    -   position 147 of the light chain is an isoleucine residue (I);    -   position 148 of the light chain is a lysine residue (K); and/or    -   position 149 of the light chain is a arginine residue (R),        according to AHo numbering.

The antibody disclosed herein may either be a full-length immunoglobulinor an antibody fragment such as, but not limited to, a Fab, a Fab′, aF(ab)′₂, a scFv, a Fv fragment, a nanobody, a VHH or a minimalrecognition unit.

In one embodiment, the antibody is monovalent, such as a scFv or a Fabfragment. In another embodiment, the antibody is multivalent. Suchmultivalent molecule can be bivalent (such as a full-lengthimmunoglobulin or a F(ab′)₂ fragment) or comprises at least three targetbinding sites. The multivalent antibody can be a bispecific antibodysuch as a diabody, a single-chain diabody or a tandem scFv (see, e.g.,KONTERMANN, R. E. Methods in Molecular Biology. Edited by LO, B. Totowa,N.J.: Humana Press, 2004. ISBN 1588290921. p. 227-242). Said bispecificantibodies may well use shorter linkers then SEQ ID NO: 14, i.e., havingonly one to three repeats of the basic motif of SEQ ID NO: 14 (see,e.g., HOLLIGER, P., et al. Diabodies: small bivalent and bispecificantibody fragments. PNAS 1993, vol. 90, no. 14, p. 6444-6448). Inanother embodiment, the multivalent antibody is a triabody, minibody ortetrabody.

In a preferred embodiment, the antibody and in particular the monovalentantibody fragment above is a scFv. The VH and VL domains can beconnected in either orientation; VL-linker-VH or VH-linker-VL, by aflexible linker. In a preferred embodiment, the orientation isVL-linker-VH, I.e. with the light chain variable region at theN-terminal end and the heavy chain variable region at the C-terminal endof the polypeptide.

Such antibody, and in particular the monovalent antibody fragment suchas the scFv, is stable. As used herein, the term “stable” refers to thebiophysical property of the antibody to remain essentially monomeric insolution after prolonged incubation and/or incubation at elevatedtemperature.

For example, the antibodies provided herein and in particular themonovalent antibody fragment above, more particularly the scFv, remainmonomeric at least to 70%, preferably at least to 75%, and mostpreferably to 80% after being incubated for 1 month at 37° C. at aconcentration of 1 mg/ml in PBS at pH 7.2. Additionally oralternatively, the antibody remains monomeric at least to 80%,preferably at least to 85%, more preferably to 90%, after 1 month atroom temperature at a concentration of 1 mg/ml in PBS at pH 7.2.

The percentage of monomers can, e.g., be determined by SEC-HPLC (sizeexclusion chromatography-high-performance liquid chromatography). Asuitable mobile phase for such testing is, e.g., PBS at pH 7.2. Themonomer content can be quantified by peak integration of the UV280 nmsignal recorded during the protein chromatography. A suitable HPLCsystem is, e.g., a Dionex Summit HPLC controlled by Chromeleon® 6.5software that also allows subsequent chromatogram analysis and peakquantification.

In a preferred embodiment, the antibodies provided herein, and inparticular the antibody fragment above, comprise a VH domain of humansubtype VH3 or VH1b, preferably of subtype VH3. As known in the art, VHdomains of other human subtypes, i.e. VH2, VH4 and VH6, usually areunstable in the scFv format which is, e.g., reflected by a lowercooperativity in equilibrium unfolding than VH3 or VH1b (see, e.g.EWERT, S. et al. Structure-based improvement of the biophysicalproperties of immunoglobulin VH domains with a generalizable approach.Biochemistry 2003, vol. 42, p. 1517-1528). Indeed, a library of scFvderived from naturally occurring human full-length immunoglobulins beingselected for stability and solubility within yeast cells showed apreference for VH3 (67%) and VH1b (19%), whereas only 9% of VH1a and 5%of VH4 were found; VH2, VH5 and VH6 were not represented at all(WO03/097697, ESBATech AG).

) The antibodies disclosed herein are preferably cross-reactive withcynomolgous, rhesus macaque and/or mouse IL-1 beta.

The antibodies provided herein, in particular the antibody fragmentabove, comprise a variable light chain sequence having at least 90%sequence identity, more preferably at least 94%, 95%, 96%, 97%, 98% or99% sequence identity to SEQ ID Nos: 7, 8 or 24; and a variable heavychain sequence having at least 90% sequence identity, more preferably atleast 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID Nos: 9,10, 11, 12 or 13.

In one embodiment, the antibodies disclosed herein comprise a variablelight chain sequence having at least 90% sequence similarity, morepreferably at least 94%, 95%, 96%, 97%, 98% or 99% sequence similarity,to SEQ ID Nos: 7, 8 or 24; and a variable heavy chain sequence having atleast 90% sequence similarity, more preferably at least 94%, 95%, 96%,97%, 98% or 99% sequence similarity, to SEQ ID Nos: 9, 10, 11, 12 or 13.

The antibodies provided herein preferably comprise a VL sequence as setforth in SEQ ID Nos. 7, 8 or 24. Additionally or alternatively, theantibody comprises a VH as set forth in SEQ ID Nos. 9, 10, 11, 12 or 13.

The antibody, in particular in case of a scFv, may comprise a linkersequence. Such linker sequence has typically ten to about 25 aminoacids. Usually, such linker peptide is rich in glycines, which conferflexibility, as well as serines and/or threonines for improvedsolubility. In a preferred embodiment, a (GGGGS)₄ linker (SEQ ID NO: 14)or a variant thereof is used. Variations of said motif having three tofive repeats may also be used. Further suitable linkers are described,e.g., in ALFTHAN, K. Properties of a single-chain antibody containingdifferent linker peptides. Protein Engineering 1995, vol. 8, no. 7, p.725-731.

In one embodiment, the antibody comprises the sequence as set forth inSEQ ID No.: 16, 17, 18, 19 or 20. In a much preferred embodiment, theantibody comprises the sequence as set forth in SEQ ID No.: 19.

In certain embodiments, variants of the antibodies provided herein arecontemplated. For example, it may be desirable to improve antigenbinding, antibody-dependent cell-mediated cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC) to increase stability orsolubility, to decrease immunogenicity and/or to alter other biological,biochemical or biophysical properties of the antibody. In someembodiments the variant does not show any improvement over the parentantibody.

Variants of the antibodies provided herein may be prepared by proteinand/or chemical engineering, introducing appropriate modifications Intothe nucleic acid sequence encoding the antibody, or by protein/peptidesynthesis, Any combination(s) of deletions, substitutions, additions andinsertions can be made to the framework or to the CDRs, provided thatthe generated antibody possesses the desired characteristics for whichit can be screened using appropriate methods. Of particular interest aresubstitutions, preferably conservative substitutions as described above.Preferred conservative substitutions include:

1. Substituting alanine (A) by valine (V);

2. Substituting arginine (R) by lysine (K);

3. Substituting asparagine (N) by glutamine (Q);

4. Substituting aspartic acid (D) by glutamic acid (E);

5. Substituting cysteine (C) by serine (S);

6. Substituting glycine (G) by alanine (A);

7. Substituting histidine (H) by arginine (R) or lysine (K);

8. Substituting isoleucine (I) by leucine (L);

9. Substituting methionine (M) by leucine (L);

10. Substituting phenylalanine (F) by tyrosine (Y);

11. Substituting proline (P) by alanine (A);

12. Substituting serine (S) by threonine (T);

13. Substituting tryptophan (W) by tyrosine (Y);

14. Substituting phenylalanine (F) by tryptophan (W); and/or

15. Substituting valine (V) by leucine (L) and vice versa.

The antibody described herein may comprise one or more, such as two,three, four, five, six, seven, eight, nine, ten, eleven, twelve or moreof such conservative substitutions.

Non-conservative substitutions may lead to more substantial changes,e.g., with respect to the charge, dipole moment, size, hydrophilicity,hydrophobicity or conformation of the polypeptide. In one embodiment,the antibody comprises one or more, such as two, three, four, five, six,seven, eight, nine, ten, eleven, twelve or more of such non-conservativesubstitutions.

) A variant antibody can comprise modifications in the CDRs or in theframework sequences. For example, the CDRs provided herein may compriseone, two, three, four, five or even more modifications. In oneembodiment, the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 takenas a whole are at least 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or more preferably 99% identical to the CDRs provided herein.Additionally or alternatively, the CDR-L1, CDR-L2, CDR-L3, CDR-H1,CDR-H2 and CDR-H3 taken as a whole are at least 85%, preferably 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more preferably 99% similar tothe CDRs provided herein.

Additionally or alternatively, the VH of the antibody comprisessolubility enhancing point mutations. WO2009/155725 (ESBATech, an AlconBiomedical Research Unit LLC) describes a motif, which has proven toincrease the overall solubility of the antibody. The residues are placedat positions located in the interface of the variable domain and theconstant domain of an antibody and stabilize antibody fragments, inparticular scFv, lacking the constant domain. In particular, at leastone, preferably all three of the following residues are present:

-   -   (i) serine (S) at heavy chain amino acid position 12 (according        to AHo numbering);    -   (ii) serine (S) or threonine (T) at heavy chain amino acid        position 103 (according to AHo numbering); and/or    -   (iii) serine (S) or threonine (T) at heavy chain amino acid        position 144 (according to AHo numbering).

In a preferred embodiment, the antibody has a serine at VH position 12;a serine at VH position 103; and a threonine at VH position 144 (all AHonumbering).

In a preferred embodiment, the antibody disclosed herein comprises avariable light chain of SEQ ID No.: 8 and a variable heavy chain of SEQID No.: 12.

Variants may also be prepared by chain shuffling of light and heavychains. A single light chain can be combined with a library of heavychains to yield a library of variants. In one embodiment, said singlelight chain is selected from the group of VL sequences disclosed hereinand/or said library of heavy chains comprises one or more of the VHsequences disclosed herein. Likewise, a single heavy chain can becombined with a library of light chains. Preferably, said single heavychain is selected from the group of VH sequences recited above and/orsaid library of light chains comprises one or more of the VL sequencesrecited above.

In one embodiment, a variant VH sequence comprises a sequence selectedfrom the group consisting of SEQ ID Nos.: 9, 10, 11 and 13.

In another embodiment, a variant VL sequence comprises a sequenceselected from the group consisting of SEQ ID Nos.: 7 and 24.

Preferably, a variant antibody

(i) retains specific binding to IL-1 beta, in particular to hIL-1 beta;

(ii) has a potency (IC₅₀) with regard to inhibiting the biologicaleffect of human IL-1 beta of lower than 10 nM, preferably lower than 1nM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, more preferably oflower than 25 pM; and

(iii) competes with the antibody disclosed herein for binding to IL-1beta.

The antibodies disclosed herein have high potencies for neutralizingIL-1 beta. Such antibodies have very high inhibitory potencies againsthuman IL-1 beta with an IC₅₀ of lower than 10 nM, more preferably lowerthan about 1 nM, 500 pM, 250 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50pM, 40 pM, 30 pM, 20 pM, 10 pM and most preferably about 5 pM.

The IC₅₀ can, e.g., be determined using a cell based potency assay. Inone embodiment, the IC₅₀ value above is determined by inhibiting theIL-1 beta induced release of IL-6 from human fibroblasts. Such assay isbased on the observation that fibroblasts stimulated with IL-1 betarelease IL-6. In the presence of IL-1 beta inhibiting antibodies, theconcentration of released IL-6 is reduced. In a preferred embodiment,normal human dermal fibroblasts (NHDF-Neo, e.g., from Lonza WalkersvilleUSA, cat. no. CC-2509) cells are used. Upon incubation with a mixture ofhIL-1 beta and the antibody of interest, supernatants are harvested andexamined by an IL-6 ELISA such as the R&D Systems Human IL-6 DuoSetELISA kit (R&D Systems, cat. no. DY206). In one embodiment, the assay isthe IL-1 beta neutralization assay as described in example 2. In apreferred embodiment, the assay is the IL-1 beta neutralization assay asdescribed in example 4. The IC₅₀ value is the mean value obtained fromthree independent repetitions of such assay.

In a much preferred embodiment, the antibody has a melting temperatureof about 69° C. as determined by differential scanning fluorimetry(DSF), preferably 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76°C., 77° C., 78° C., 79° C., and most preferably 80° C. This method isbased on the properties of certain dyes being fluorescent only in ahydrophobic environment. For example, protein unfolding can be detectedas an increase in fluorescence upon binding of the dye SYPRO® Orange toa heat-denatured protein (NIESEN F. H. et al. The use of differentialscanning fluorimetry to detect ligand interactions that promote proteinstability. Nature Protocols 2007, vol. 2, p. 2212-2221). The stabilityof a protein can thus be analyzed by thermal denaturation.

In some embodiments, IL-1 beta shows residual activity when contactedwith the antibodies disclosed herein in an in vivo and/or an in vitrosetting, i.e. the antibody does not completely inhibit the action ofIL-1 beta but permits a residual activity signal elicited by IL-1 beta.This may have the advantage of maintaining efficient immune responses.In one embodiment, the antibody allows a residual IL-1 beta activity ofabout 10%, preferably of about 5% of the assay signal as determined byinhibiting the release of IL-6 from human fibroblasts by 10 pg/ml ofIL-1 beta, preferably the assay described in example 2, in the presenceof 60 ng/ml of the antibody described herein when compared to antibodiesof non-relevant specificity or vehicle control at the sameconcentration.

Also provided are biosmilars of the antibodies described herein, i.e.compounds showing no meaningful differences in terms of safety, purityand potency of the antibodies above.

Nucleic Acids, Vectors, Host Cells and Method of Production

Each antibody described herein is encoded by a single nucleic acid or bytwo or more nucleic acids, for example, each encoding at least onevariable region. Knowing the sequence of the antibody or of its parts,cDNAs encoding the polypeptide sequence can be generated by methods wellknown in the art, e.g., by gene synthesis. These cDNAs can be cloned bystandard cloning and mutagenesis techniques into a suitable vector suchas an expression vector or a cloning vector. Optionally, the variablelight chain is encoded by a separate nucleic acid than the variableheavy chain of the antibody. Further, additional sequences such as tags(e.g., a His-tag), constant domains for the production of a Fab or afull-length immunoglobulin, linkers, coding sequences of a secondbinding specificity or another functional polypeptide such as an enzymeto generate a fusion construct or a bispecific molecule may be includedinto the genetic construct.

Based on the cloning strategy chosen genetic constructs may generate anantibody having one or more additional residues at the N-terminal orC-terminal end. For example, an N-terminal methionine derived from thestart codon or an additional alanine may be present in an expressedpolypeptide, unless it has been clipped off post-translationally. It istherefore to be understood that the antibodies disclosed herein comprisethe disclosed sequences rather than consist of them.

In one embodiment, the invention provides a nucleic acid sequencecomprising at least 300 nucleobases, more preferably at least 350, 400,450, or 500 nucleobases and having at least 85%, more preferably atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to SEQ ID No. 15. In a much preferred embodiment, the nucleicacid sequence is SEQ ID No. 15.

Additionally or alternatively, the invention provides a nucleic acidsequence comprising at least 300 nucleobases, more preferably at least350, 400, 450, or 500 nucleobases, which hybridizes with the nucleicacid of SEQ ID No. 15 under high stringency conditions.

Basic protocols of standard cloning, mutagenesis and molecular biologytechniques are described in, e.g., Molecular Cloning, A LaboratoryManual (GREEN, M. and Sambrook, J. Molecular Cloning: a LaboratoryManual. 4th edition. Cold Spring Harbor Laboratory, 2012. ISBN1936113422).

Appropriate host cells for the expression of the genetic constructs canbe prokaryotic or eukaryotic. Suitable prokaryotic host cells aregram-negative or gram-positive and include species of the Escherichia,Erwinina, Enterobacter, Klebsiella, Pseudomonas or Bacillus families,Much preferred is Escherichia coli, in particular E. coli strains BL21(DE3) (Life Technologies™, cat. no. C6000-03) and Origami™ 2 (DE3)(Novagen, cat. no 71345).

If post-translational modifications such as glycosylation orphosphorylation are desired, eukaryotic host cells are preferable. Forexample, eukaryotic microbes such as commonly used Saccharomycescerevisiae or Pichia pastoris strains may serve as host cells. Hostcells can also include plant or animal cells, in particular insect ormammalian cells. Suitable mammalian cells include, without being limitedto, Chinese Hamster Ovary Cells (CHO), Human Embryonic Kidney Cells(HEK), Human Umbilical Vein Endothelial Cells (HUVEC) or NS0 myelomacells.

The antibody can be produced by expression in a suitable host cell. Forexample, the expression vectors described above are introduced into ahost cell by standard techniques such as electroporation or chemicaltransformation. The transformed cells are then cultivated underconditions adequate for recombinant protein expression, typically inappropriate nutritional media, optionally modified for inducingpromotors, selecting transformants, or amplifying encoding sequences ofinterest. The antibody protein is recovered from the culture andoptionally purified using standard techniques in the art. The yield ofrecombinant protein may be improved by optimizing media and cultureconditions such as pH, temperature or oxygen supply. In prokaryotes theantibody can be produced in the periplasm, intracellularly as inclusionbodies or be secreted into the medium. Upon harvest, the protein can bepurified using methods well known in that art such as size exclusionchromatography, ion exchange chromatography, reversed phasechromatography, hydrophobic interaction, mixed mode chromatographyand/or affinity chromatography.

In one embodiment, the antibody is produced in a cell-free system. Thistypically involves in vitro transcription followed by in vitrotranslation of nucleic acid product templates encoding the proteinsdescribed herein, e.g., plasmid DNA or PCR product templates. Forexample, crude lysates from growing cells are used as basis forcell-free expression systems, providing the necessary enzymes as well asthe cellular protein synthesis machinery. The necessary building blockssuch as amino acids or nucleobases as well as energy deliveringmolecules and others can be exogenously supplied. Cell-free expressionsystems can, for example, be based on lysed rabbit reticulocytes (e.g.,Rabbit Reticulocyte Lysate System, Promega, cat. no. L4540), HeLa cells(e.g., 1-Step Human In Vitro Translation Kit, Thermo Scientific, cat.no. 88881), insect cells (e.g., EasyXpress Insect Kit II, Qiagen, cat.no. 32561), wheat germs (e.g., Wheat Germ Extract, Promega, cat. no.L4380), or E. coli cells (e.g., PURExpress® In Vitro Protein SynthesisKit, NEB, cat. no. E6800S). Also, optimized cell-free antibodyexpression systems for improved disulfide bond generation can be usedfor production. Commercially available kits include insect cell lysates(e.g., EasyXpress Disulfide Insect Kit, Qiagen, cat. no. 32582) or E.coli cell lysates (e.g., EasyXpress Disulfide E. coli Kit, Qiagen, cat.no. 32572). Cell-free protein synthesis has, e.g., the advantage ofbeing fast, achieving high product yields, allowing for easymodification of reaction conditions, forming a low degree of or even noby-products. Cell-free protein synthesis may involve biological and/orchemical steps which cannot be conducted in purely biological orchemical production systems. For example, non-natural orchemically-modified amino acids can be incorporated into the protein atdesired positions. ScFv-toxin fusion proteins have been successfullyproduced in cell-free systems (NICHOLLS, P. J., et al. Characterizationof single-chain antibody (sFv)-toxin fusion proteins produced in vitroin rabbit reticulocyte lysate. Journal of Biological Chemistry 1993,vol. 268, pp. 5302-5308.) Thus, in one embodiment, a method of producingthe antibody described herein is provided comprising the steps of

-   -   (a) providing a cell-free system,    -   (b) providing a nucleic acid template encoding the antibody        described herein,    -   (c) allowing for transcription and translation of said nucleic        acid product template, whereby the antibody is expressed;    -   (d) recovering the antibody; and optionally    -   (e) purifying said antibody.

Additionally or alternatively, a method of producing the antibodydescribed herein comprises at least one step of chemical synthesis. Forexample, the method may be entirely chemical. In another embodiment, thecell-based or the cell-free production systems described above compriseat least one step of chemical synthesis.

In a preferred embodiment, the antibodies described herein are producedin a cell-based system using an expression vector for intracellularexpression in E. coli. Upon expression the polypeptide is generated asinclusion bodies within the cells which are subsequently separated fromfurther cell particles followed by solubilisation in a denaturing agentsuch as guanidine hydrochloride (GndHCl) and refolded by renaturationprocedures well known to the skilled person.

Chemical and/or Biological Modifications

In one aspect, the antibody of the instant invention is chemicallyand/or biologically modified. Such modification may comprise, but is notlimited to, glycosylation, PEGylation, HESylation, Albumin fusiontechnology, PASylation, labelling with dyes and/or radioisotopes,conjugation with enzymes and/or toxins, phosphorylation, hydroxylationand/or sulfation. Likewise, the nucleic acid sequence, the vector and/orthe host cell described above can be modified accordingly.

Chemical and/or biological modifications may be conducted, e.g., tooptimize pharmacokinetics, pharmacodynamics, water solubility of theprotein and/or to lower its side effects. For example, PEGylation,PASylation and/or HESylation may be applied to slow down renal clearanceand thereby increase plasma half-life time of the antibody. Additionallyor alternatively, a modification may add a different functionality tothe protein, e.g., the antibody may be conjugated with a toxin to moreefficiently combat cancer cells, or a detection molecule may be addedfor diagnostic purposes.

Glycosylation refers to a process that attaches carbohydrates toproteins. In biological systems, this process is performed enzymaticallywithin the cell as a form of co-translational and/or post-translationalmodification. A protein, here the antibody, can also be chemicallyglycosylated. Typically, but not limited to, glycosylation is (i)N-linked to a nitrogen of asparagine or arginine side-chains; (ii)O-linked to the hydroxy oxygen of serine, threonine, tyrosine,hydroxylysine, or hydroxyproline side-chains; (iii) involves theattachment of xylose, fucose, mannose, and/or N-acetylglucosamine to aphospho-serine; or (iv) in form of C-mannosylation wherein a mannosesugar is added to a tryptophan residue found in a specific recognitionsequence. Glycosylation patterns can, e.g., be controlled by choosingappropriate cell lines, culturing media, protein engineeringmanufacturing modes and/or process strategies (HOSSLER, P. Optimal andconsistent protein glycosylation in mammalian cell culture. Glycobiology2009, vol. 19, no. 9, p. 936-949).

Protein engineering to control or alter the glycosylation pattern mayinvolve the deletion and/or the addition of one or more glycosylationsites. The creation of glycosylation sites can conveniently beaccomplished by introducing the corresponding enzymatic recognitionsequence into the amino acid sequence of the antibody or by adding orsubstituting one or more of the above enumerated amino acid residues.

It may be desirable to PEGylate the antibody. PEGylation may alter thepharmacodynamic and pharmacokinetic properties of a protein.Polyethylene-glycol (PEG) of an appropriate molecular weight iscovalently attached to the protein backbone (see, e.g., PASUT, G. andVeronese, F. State of the art in PEGylation: the great versatilityachieved after forty years of research. Journal of Controlled Release2012, vol. 161, no. 2, p. 461-472). PEGylation may additionally reducethe immunogenicity by shielding the PEGylated protein from the immunesystem and/or alter its pharmacokinetics by, e.g. increasing the in vivostability of the antibody, protecting it from proteolytic degradation,extending its half-life time and/or by altering its biodistribution.

Similar effects may be achieved by PEG-mimetics, e.g., by HESylating orPASylating the antibody. HESylation utilises hydroxyethyl starch (“HES”)derivatives, whereas during PASylation the antibody becomes linked toconformationally disordered polypeptide sequences composed of the aminoacids proline, alanine and serine. Said PEG-mimetics and relatedcompounds are, e.g., described in BINDER, U. and Skerra, A. Half-LifeExtension of Therapeutic Proteins via Genetic Fusion to Recombinant PEGMimetics, in Therapeutic Proteins: Strategies to Modulate Their PlasmaHalf-Lives. Edited by KONTERMANN, R., Weinheim, Germany: Wiley-VCH,2012. ISBN: 9783527328499. p. 63-81.

The antibody may include a salvage receptor binding epitope. Suchsalvage receptor binding epitope typically refers to an epitope of theFc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) and hasthe effect of increasing the in vivo half-life of the molecule.

Additionally or alternatively, the antibody is labelled with orconjugated to a second moiety which ascribes ancillary functionsfollowing target binding. Said second moiety may, e.g., have anadditional immunological effector function, be effective in drugtargeting or useful for detection. The second moiety can, e.g., bechemically linked or fused genetically to the antibody using knownmethods in the art.

Molecules which may serve as second moiety include, without beinglimited to, radionuclides, also called radioisotopes (e.g., 35S 32P,14C, 18F, 125I); apoenzymes; enzymes (such as alkaline phosphatase,horseradish peroxidase, beta-galactosidase or angiogenin); co-factors;peptides (e.g., HIS-tags); proteins (incl. lectins, metal-bindingdomains); carbohydrates (incl, mannose-6-phosphate tag); fluorophores(including fluorescein isothiocyanate (FITC); phycoerythrin;green/blue/red and other fluorescent proteins; allophycocyanin (APC));chromophores; vitamins (including biotin); chelators; antimetabolites(e.g., methotrexate), liposomes; toxins including cytotoxic drugs suchas taxol, gramicidin D or colchicine; or a radiotoxin.

A labelled antibody is particularly useful for in vitro and in vivodetection or diagnostic purposes. For example, an antibody labelled witha suitable radioisotope, enzyme, fluorophore and/or chromophore can bedetected by radioimmunoassay (RIA), enzyme-linked immunosorbent assay(ELISA), or flow cytometry-based single cell analysis (e.g., FACSanalysis). Similarly, the nucleic acids and/or vectors disclosed hereincan be used for detection or diagnostic purposes, e.g., using labelledfragments thereof as probes In hybridization assays. Labelling protocolsmay, e.g., be found in JOHNSON, I. and Spence, M. T. Z. Molecular ProbesHandbook, A Guide to Fluorescent Probes and Labeling Technologies. LifeTechnologies, 2010. ISBN: 0982927916.

Compositions

The antibody of the instant invention, the nucleic acid sequences or thevector disclosed herein can be provided in a composition which furthercomprises a suitable carrier, excipient or diluent. Much preferred is acomposition comprising an antibody described herein.

Such composition can, e.g., be a diagnostic, a cosmetic or apharmaceutical composition. For therapeutic or cosmetic purposes, saidcomposition is a pharmaceutical composition comprising an effectiveamount of antibody described herein and further a pharmaceuticalcarrier, excipient or diluent, i.e. a carrier, excipient or diluent notbeing toxic at the dosages and a concentration employed.

Suitable “carrier”, “excipients” or “diluents” include, without beinglimited to: (i) buffers such as phosphate, or organic acids such ascitrate; (ii) antioxidants such as ascorbic acid and tocopherol; (iii)preservatives such as 3-pentanol, hexamethonium chloride, benzalkoniumchloride, benzyl alcohol, alkyl paraben, catechol, or cyclohexanol; (iv)amino acids, such as e.g. histidine, arginine; (v) peptides, preferablyup to 10 residues such as polylysine; (vi) proteins, such as bovine orhuman serum albumin; (vii) hydrophilic polymers such aspolyvinylpyrrolidone; (viii) monosaccharides, disaccharides,polysaccharides and/or other carbohydrates including glucose, mannose,sucrose, mannitol, trehalose, sorbitol, aminodextran or polyamidoamines;(ix) chelating agents, e.g., EDTA or EGTA; (x) salt-forming ions such assodium or potassium; (xi) metal complexes (e.g., Zn-protein complexes);and/or (xii) ionic and non-ionic surfactants such as TWEEN™, PLURONICS™or polyethylene glycol (PEG).

Many of said exemplary compounds have different and/or sometimes dual ormultiple functions and may, e.g., act as carrier and as diluent. It isalso to be understood that the composition may comprise more than one ofeach carrier, diluent or excipient.

The antibody, the nucleic acid sequences or the vector may be providedon solid support materials such as beads and microparticles, Typically,the molecules are linked to such carrier via a covalent bond (optionallyinvolving a linker), but may also non-covalently adhere to such carrieror admixture. Said beads and microparticles can comprise, for example,starch, cellulose, polyacrylate, polylacetate polyglycolate,poly(lactide-co-glycolide), latex, or dextran.

Therapeutic Applications

The molecules described herein, in particular the antibody, bindingmember, nucleic acid or vector, are useful as a medicament. Typically,such medicament comprises a therapeutically effective amount of themolecules provided herein. Accordingly, said molecules can be used forthe production of a medicament useful in the treatment of IL-1beta-related disorders.

In one aspect, a method of treating an IL-1 beta-related disorder isprovided comprising the steps of administering a pharmaceuticallyeffective amount of the molecules described herein, in particular theantibody, to a subject in need thereof. In one embodiment, thepharmaceutical composition above comprising such pharmaceuticallyeffective amount of the antibody is administered to said subject.

The term “treat” or “treatment” as used herein refers to theadministration of a pharmaceutically effective amount of the antibody,nucleic acid, vector or host cell of the instant-invention, to a subjectin need thereof to prevent, cure, delay the onset and/or progression,reduce the severity of, stabilize, modulate, cure or ameliorate one ormore signs and/or symptoms of an IL-1 beta-related disorder. Typically,the antibody, nucleic acid, vector or host cell is provided in apharmaceutical composition including those previously described herein.

A “therapeutically effective amount” refers to an amount at which thedosage regimen applied yields the desired therapeutic effect, i.e., toreach treatment goals as defined above. The dosage will depend onvarious factors including patient and clinical factors (e.g., age,weight, gender, clinical history of the patient, severity of thedisorder and/or response to the treatment), the nature of the disorderbeing treated, the particular composition to be administered, the routeof administration, and other factors.

The subject in need of such treatment can be a human or a non-humananimal, e.g., a mouse, rat, rabbit, monkey, dog, horse, cow, chicken,guinea pig or pig. Typically, the subject is diagnosed with an IL-1beta-related disorder or may acquire such a disorder.

Examples of IL-1 beta-related disorders, in which antagonist of IL-1beta have shown therapeutic effects include, without being limited to,proliferative diabetic retinopathy, gouty arthritis, Schnitzlersyndrome, systemic juvenile idiopathic arthritis, rheumatoid arthritis,acute gouty arthritis, chronic gouty arthritis, urticaria, vasculitis,type 1 diabetes, type 2 diabetes, ankylosing spondylitis, recurrentmultifocal osteomyelitis, relapsing polychondritis, cyropyrin-associatedperiodic syndrome (CAPS), Behçet's disease, familial mediterraneanfever, chronic obstructive pulmonary disease, polymyalgia rheumatic,NALP3-mutations, pyoderma gangrenosum, chronic idiopathic urticarial,osteoarthritis, wet age-related macular degeneration, dry eye syndrome,pustular psoriasis, synovitis-acne-pustulosis-hyperostosis-osteitissyndrome, macrophage activation syndrome, periodicfever-adenitis-pharyngitis-aphthous ulcer syndrome, adult-onset Still'sdisease, mevalonate kinase deficiency, atherosclerosis, TNF-receptorassociated periodic syndrome (TRAPS), acne vulgaris and/or acne inversa.

The term “CAPS” or cryopyrin-associated periodic syndrome is to beunderstood to include each of familial cold autoinflammatory syndrome(FCAS), Muckle-Wells syndrome (MWS) and neonatal-onset multisysteminflammatory disease, also known as chronic infantile neurological,cutaneous and articular (CINCA) syndrome.

The pharmaceutical composition may be applied by differentadministration routes. Administration can be conducted, for example, butnot limited to, parenterally, e.g., intramuscularly, subcutaneously,epicutaneously, intravenously as a bolus or by continuous infusion,intraarticularly, intrasynovially, intracerebrally,intracerebrospinally, intrathecally, epidurally, or intraperitoneally;orally; rectally; urogenitally; topically, e.g., to the skin or the eye;intravitreally; intraocularly; oticly; intranasally; by inhalation;dermally such as intradermally or transdermally; sublingually; buccally,for example. Preferred are the topical, rectal, intranasal, intravenousand/or intradermal routes of administration.

The antibody of the instant invention, the nucleic acid sequences, thevector or host cell can be combined with one or more furthertherapeutically effective compound. Said compound may either be capableof disrupting signalling via the IL-1 receptor, or alternatively inhibitone or more different targets such as, e.g., other mediators ofinflammatory responses. Such compound(s) can be administeredsimultaneously or sequentially.

For therapeutic applications, the antibody may also be radiolabelled orlinked to a toxin or linked to another effector function as describedabove.

Diagnostic Applications and/or Detection Purposes

The antibody of the instant invention may be used for detection ordiagnostic purposes in vivo and/or in vitro. For example, a wide rangeof immunoassays involving antibodies for detecting the expression inspecific cells or tissues are known to the skilled person. Likewise, thenucleic acid sequence, the vector and/or the host cell describedpreviously can be used accordingly as detailed in this section. In oneembodiment, the method is not practised on the human or animal body.

For such applications the antibody, the nucleic acid sequence, thevector or the host cell disclosed herein may be either labelled orunlabelled. E.g., an unlabelled antibody may be used and detected by asecondary antibody recognizing an epitope on the antibody describedherein.

In another embodiment, the antibody, nucleic acid sequence, vectorand/or host cell is conjugated with one or more substances which can berecognized by a detector substance(s), e.g., the antibody beingconjugated with biotin which can be detected by streptavidin. Likewise,the nucleic acids and/or vectors disclosed herein can be used fordetection or diagnostic purposes, e.g., by using labelled fragmentsthereof as probes in hybridization assays.

In certain embodiments, any of the molecules provided herein, inparticular the antibody, is useful for detecting the presence of IL-1beta, preferably including full-length IL-1 beta, fragments thereofand/or precursors thereof, in a sample, preferably biological sample.The term “detecting” encompasses quantitative and/or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue from human patients. Non limiting examples of biologicalsamples include blood, urine, cerebrospinal fluid, biopsy, lymph and/ornon-blood tissues.

In certain embodiments, the method comprises contacting the biologicalsample with an anti-IL-1 beta-antibody as described herein underconditions permissive for binding of the antibody to IL-1 beta anddetecting whether a complex is formed between the antibody and IL-1beta. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-IL-1 beta antibody is used to select subjectseligible for therapy with the antibody described herein, e.g., whereIL-1 beta is a biomarker for selection of patients.

In another aspect, the antibody is used in cosmetic applications, e.g.,for improving the aesthetic appearance of skin.

In a further aspect, a kit is provided comprising the antibody, apackaged combination of reagents with instructions for performing thedetection or diagnostic assay. The reagents are typically provided inpredetermined amounts of dry powders, usually lyophilized, includingexcipients which after dissolution will provide a reagent solutionhaving the appropriate concentration. Other additives such asstabilizers and/or buffers may also be included. If the antibody islabelled with an enzyme, the kit will typically include the accordingsubstrates and cofactors. Likewise, any binding member, the nucleic acidsequence, the vector and/or the host cell described previously can beused accordingly as detailed in this section.

EXAMPLES Example 1—Design of Anti-Human IL-1 Beta Antibodies

Variable heavy and light chains of AB7 were linked by the peptide linkerof SEQ ID No.: 14 to yield a scFv which turned out to be unstable.

CDRs of the anti-human IL-1 beta antibody comprising SEQ ID No.: 22 andSEQ ID No.: 23 were identified and grafted onto two stable human scFvframeworks, each consisting of an N-terminal human light chain variableregion, a linker sequence and a C-terminal human heavy chain variableregion. Both scFv frameworks differ from each other in five amino acidpositions in the heavy chain. The resulting two scFvs were termedDLX2260 and DLX2289 (see FIG. 1 ).

The DLX2289 framework is derived from a human immunoglobulin describedin WO 03/097697 A (ESBATech AG), whereas the DLX2260 framework isderived thereof; the VH framework sequence of the latter has beenmodified for humanization and stabilization of rabbit antibodies, see,e.g., WO 2009/155726 A (ESBATech, AN ALCON BIOMEDICAL RESEARCH UNIT LLC;BORRAS, L., et al. Generic approach for the generation of stablehumanized single-chain Fv fragments from rabbit monoclonal antibodies.Journal of Biological Chemistry 2010, vol. 285, no. 12, p. 9054-9066).

The scFvs were expressed as inclusion bodies in E. coli (BL21 (DE3);Novagen, cat. no. 69450-3). After harvesting the cells the inclusionbodies were isolated and purified. The proteins were refolded bystandard methods and monomeric scFvs were purified using size exclusionchromatography. Monomeric peak fractions were collected andcharacterized for antigen binding and IL-1 beta neutralization potencyin a cell-based assay.

Example 2—IL-1 Beta Binding and Neutralization by DLX2260 and DLX2289

In an ELISA, the recognition of recombinant human (rh) IL-1 beta byDLX2260 and DLX2289 was confirmed. The scFvs were coated on Maxisorp96-well microplates at a concentration of 10 mcg/ml overnight at 4° C.in 50 mM Glycine, 50 mM NaCl, pH 10.0. After blocking with 5% non-fatdry milk, biotinylated rhIL-1 beta (R&D systems, cat. no. NFLB0) wasadded at concentrations ranging from 5 to 20 ng/ml. Bound IL-1 beta wasdetected by Streptavidin-HRP (BD Pharmingen, cat. no. 554060). The ELISAwas developed with BM Blue POD substrate (Roche Applied Science) and theabsorbance was measured at 450 nm. The results (see FIG. 2A) show thatboth scFvs bind equally well to rhIL-1 beta.

The potency of DLX2260 and DLX2289 to neutralize the biological functionof human IL-1 beta was analysed in a cell proliferation assay usingD10.G4.1 mouse T helper cells (DSMZ, cat. no. ACC45). D10.G4.1 cellswere seeded in 96-well tissue culture plates at 4′000 cells per well inRPMI 1640 w/o phenolred (Gibco, Life Technologies, cat. no. 32404014)supplemented with 10% FBS (Gibco, Life Technologies, cat. no. 10270106),2 mM glutamine (Gibco, Life Technologies, cat. no. 25030024), 1%PenStrep (Gibco, Life Technologies, cat. no. 15070063) and 10 ng/ml ofrhIL-2 (Peprotech, cat. no. 200-02), and cultured for 4-6 hours. ThescFvs and control antibody (R&D systems, cat. no. MAB201) werepre-incubated with rhIL-1 beta (Peprotech, cat. no. 200-01B) in cellculture media for 2 hours at 37° C. The mixtures were added to the cellsat a final concentration of 10 pg/ml of rhIL-1 beta. The positiveproliferation control contained 10 pg/ml of rhIL-1 beta only. The cellswere cultured for an additional 48 hours. IL-1 beta-inducedproliferation was quantified by addition of XTT (Sigma-Aldrich, cat. no.X4251-100MG) at a concentration of 1 mg/ml in RPMI w/o phenolred with 25mcM phenazine methosulfate (Sigma-Aldrich, P9625-1G). The cells wereincubated with XTT solution for 4 hours at 37° C. Absorbance wasdetermined at 450 nm and at the reference wavelength of 620 nm. BothDLX2260 and DLX2289 inhibited IL-1 beta-induced cell proliferation (seeFIG. 2B) with similar efficiency. Compared to the control full-lengthimmunoglobulin MAB201 (R&D systems, cat. no. MAB201), cell proliferationis not inhibited completely, approximately 5% of cells continued growingeven at highest concentrations of scFvs.

Example 3—Engineering of DLX2260

DLX2260 was chosen as starting point for framework mutations to improveits capability of human IL-1 beta neutralization. Several mutations wereintroduced in the light chain as well as the heavy chain frameworks. Oneposition in CDR-H2 was also mutated. Additionally, three amino acidpositions in the heavy chain framework that were known to improvesolubility of scFvs (see WO2009/155725, ESBATech, an Alcon BiomedicalResearch Unit LLC) were substituted by amino acids with more hydrophilicside chains. In total, three different scFvs were designed (see FIG. 1). DLX2332 is 97% identical to DLX2260, whereas DLX2296 is 93% identicalto DLX2260. DLX2295 contains the same mutations as DLX2296 but includesin addition the three solubility substitutions as described above.DLX2295 is 92% identical to DLX2260.

DNA sequences encoding the three scFvs were cloned into a bacterialexpression vector, proteins were expressed and purified as describedabove. Monomeric scFvs were characterized for their IL-1 beta binding,neutralization and stability characteristics.

Example 4—IL-1 Beta Binding and Neutralization by DLX2295, DLX2296 andDLX2332

The recognition of rhIL-1 beta by DLX2295, DLX2296 and DLX2332 wasconfirmed by ELISA (FIGS. 3A and 3B). A scFv of irrelevant specificitywas used as a control. rhIL-1 beta (Peprotech, cat. no. 200-01B) wascoated to NUNC 96-well Maxisorp immunoplates. After blocking scFvs weretitrated from 10 to 3′000 ng/ml. Bound scFvs were detected by ProteinL-HRP (Sigma-Aldrich, cat. no. P3226), and the ELISA was developed usingBM Blue POD substrate (Roche Applied Science, cat. no. 11484281001). Forquantification purposes, the absorption was measured at 450 nm usingVersaMax microplate reader (Molecular Devices).

A fibroblast assay was performed to determine the potency of a compoundto neutralize IL-1 beta activity. Human dermal fibroblasts (NHDF-Neo,cat. no. CC-2509, Lonza Walkersville, USA), upon activation by IL-1beta, specifically release IL-6 which is quantifiable by ELISA®Neutralization of IL-1 beta decreases the amount of IL-6 released fromsuch fibroblasts. The inhibitory potency of anti-IL-1 beta antibodies isusually quantified by measuring the half-maximal reduction (IC₅₀) ofIL-1 beta-induced IL-6 release. Human dermal fibroblasts were seeded in96-well microplates at 5000 cells/well about 16-20 hours prior toaddition of samples containing the stimulus IL-1 beta. The fibroblastswere cultured in fibroblast basal medium (FBM; Lonza, cat. no. CC-3131)with supplements (hFGF-B, insulin, FBS, GA-1000) as described by thecell supplier (Lonza Walkersville, USA: Clonetics™ Dermal FibroblastCell Systems).

) Before seeding, FBM was removed and cells were washed with Dulbecco'sModified Eagle Medium (DMEM; Gibco, Life Technologies, cat. no. 11880)to remove growth factors. The cells were then cultured for 7 hours inDMEM media supplemented with 2% FBS. Antibodies were pre-incubated inDMEM media supplemented with 2% FBS with rhIL-1 beta for 1 hour at 37°C. The mixture was added to the cells at a final concentration of 10pg/ml of IL-1 beta. As a control, 10 pg/ml of rhIL-1 beta was added tocells without any anti-IL-1 beta antibody. The cells were incubated withthe IL-1 beta/anti-IL-1 beta antibody mixture for 18-24 hours, and cellculture supernatants were analyzed for IL-6 release using the Human IL-6DuoSet ELISA Kit according to the manufacturer's instructions (R&DSystems, USA, cat. no. DY206).

DLX2260, DLX2295, DLX2296 and DLX2332 were assessed for their IL-1 betaneutralization capacity in the human dermal fibroblast assay (NHDF-Neo,cat. no. CC-2509, Lonza Walkersville, USA) as described above. Asfurther controls, anti-IL-1 beta full-length immunoglobulins such asMAB201 (R&D systems, USA, cat. no. MAB201) and Canakinumab (Novartis,Ilaris®) were used. In one experiment, MAB201, DLX2296 and DLX2260 werecompared (FIG. 4 ). In another experimental series, MAB201, DLX2295 andCanakinumab were assayed in parallel (FIG. 5 ). Finally, theneutralizing efficacy of DLX2295 and DLX2332 were compared (see FIG. 6). MAB201 and DLX2295 displayed the lowest IC₅₀ both with approximately2-5 pM, whereas DLX2296 showed an IC₅₀ (considering 20% residualactivity) of about 8 pM and 2260 an IC₅₀ (considering 20% residualactivity) of about 100 pM. The IC₅₀ values are mean values of threeindependent experiments. The monovalent scFv DLX2295 is thus as potentas the bivalent MAB201 IgG. For all scFvs (DLX2295, DLX2296, DLX2260 andDLX2332) tested in the fibroblast assay residual activity was observed,i.e., the IL-1 beta activity was not inhibited completely. Thisobservation is consistent with the results obtained in the D10.G4.1proliferation assay.

Example 5—Stability

DLX2295 protein stability at different temperatures was assessed. Tothis end, HPLC (Dionex, Summit system) size exclusion chromatography(Tosoh, TSKgel G2000SWxI, cat. no. 08540) was deployed to determine thepercentage of monomeric, non-degraded scFv protein after one month at RTin PBS pH 7.2. The percentage of monomer was measured at the startingpoint of the study (T0) and after one month for 0.6 mg/ml of DLX2295.The results of the stability study are listed in table 1.

TABLE 1 monomer content DLX2295, T0 98% DLX2295, stored at RT for 1month 93%

The thermal stability of DLX2260, DLX2289, DLX2295, DLX2296 and DLX2332was analysed by differential scanning fluorimetry (DSF). For thismeasurement a real-time PCR device (Corbett, Rotor-Gene) was used toheat the scFvs in a temperature gradient while simultaneously measuringthe fluorescence. The samples contained 0.5 mg/ml of scFv and 20×SYPRO®Orange (Sigma-Aldrich, cat. no. S5692, 5000x) in PBS pH 7.2. Thetemperature gradient was set from 30° C. to 95° C. (raising in 1° C.steps, holding time of 5 seconds per step). The fluorescence was excitedat 470 nm, the emission detected at 555 nm. The midpoint meltingtemperatures (Tm) were calculated using Rotor-Gene 6000 Series Software1.7. Tm of the 5 scFvs are summarized in table 2.

TABLE 2 Tm DLX2260 79° C. DLX2289 69° C. DLX2332 73° C. DLX2296 71° C.DLX2295 70° C.

Example 6—Cross-Reactivity of DLX2295

Cross-reactivity of DLX2295 to IL-1 beta homologs from other speciesthan human beings was assessed in ELISA. Binding to the recombinantlyexpressed IL-1, beta proteins of the following species was investigated:cynomolgus (Sino Biological Inc., USA, cat. no. 90010-CNAE), rhesusmacaque (R&D Systems, USA, cat. no. 1318-RL/CF) and mouse IL-1 beta(BioLegend, cat, no. 575102). Binding of DLX2295 was compared toELISA-positive control antibodies (R&D Systems, USA, goat anti-humanIL-1 beta polyclonal IgG, cat. no. AB-201-NA; BioLegend, Inc., USA,biotin anti-mouse/rat IL-1 beta antibody, cat. no. 503505). Briefly,proteins were coated at a concentration of 2 mcg/ml over night at 4° C.on Maxisorp 96-well microplates in PBS pH 7.2. After blocking with 5%non-fat dry milk, Increasing concentrations (0.1 mcg/ml, 0.3 mcg/ml and1.0 mcg/ml) of DLX2295 were added to the wells. Successful coating ofevery protein was separately confirmed exploiting the IL-1 beta-specificcontrol antibodies. Whereas DLX2295 was detected by Protein L-HRP(Sigma-Aldrich, USA, cat. no. P3226), the control antibodies weredetected by either rabbit anti-goat IgG-HRP (Southern Biotech, cat. no.6160-05) or by Streptavidin-HRP (BD Pharmingen, USA, cat. no. 554060).The ELISAs were developed with BM Blue POD substrate (Roche AppliedScience) and the absorbance was measured at 450 nm. DLX2295 recognizedequally well human, cymonolgus and rhesus macaque IL-1 beta, while therecognition of mouse IL-1 beta was weaker.

SEQUENCE LISTING

Sequence listing The sequences disclosed herein are: VL CDR1SEQ ID No: 1 RASQDISNYLS VL CDR2 SEQ ID No: 2 YTSKLHS VL CDR3SEQ ID No: 3 LQGKMLPWT VH CDR1 SEQ ID No: 4 FSLSTSGMGVG VH CDR2SEQ ID No: 5 HIWWDGDESYNPSLKS VH CDR3 SEQ ID No: 6 NRYDPPWFVDVL of DLX2260 and DLX2289 SEQ ID No: 7EIVMTQSPSTLSASVGDRVIITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCLQGKMLPWTFGQ GTKLTVLGVL of DLX2295 and DLX2296 SEQ ID No: 8DIQMTQSPSTLSASVGDRVTITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPEDFATYFCLQGKMLPWTFGQ GTKLEIKRVH of DLX2289 SEQ ID No: 9EVQLVESGGGLVQPGGSLRLSCAASGFSLSTSGMGVGWVRQAPGKGLEWVSHIWWDGDESYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKN RYDPPWFVDWGQGTLVTVSSVH of DLX2260 SEQ ID No: 10EVQLVESGGGLVQPGGSLRLSCTASGFSLSTSGMGVGWVRQAPGKGLEWVGHIWWDGDESYNPSLKSRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARN RYDPPWFVDWGQGTLVTVSSVH of DLX2296 SEQ ID No: 11EVQLVESGGGLVQPGGSLRLSCAFSGFSLSTSGMGVGWIRQAPGKGLEWVSHIWWDGDESYNPSLKGRFTISKDTSRNTVYLQMNSLRAEDTAVYFCARN RYDPPWFVDWGQGTLVTVSSVH of DLX2295 SEQ ID No: 12EVQLVESGGGSVQPGGSLRLSCAFSGFSLSTSGMGVGWIRQAPGKGLEWVSHIWWDGDESYNPSLKGRFTISKDTSRNTVYLQMNSLRAEDTASYFCARN RYDPPWFVDWGQGTTVTVSSVH of DLX2332 SEQ ID No: 13EVQLVESGGGSVQPGGSLRLSCAASGFSLSTSGMGVGWVRQAPGKGLEWVGHIWWDGDESYNPSLKSRFTISRDTSKNTVYLQMNSLRAEDTASYFCARN RYDPPWFVDWGQGTTVTVSSlinker SEQ ID No: 14 GGGGSGGGGSGGGGSGGGGSnucleic acid sequence of DLX2295 SEQ ID No: 15GACATTCAGATGACGCAGTCTCCGTCTACCCTGTCCGCAAGTGTGGGTGATCGCGTGACAATCACCTGTCGTGCCTCACAGGACATTTCCAACTACCTGTCCTGGTATCAACAGAAACCGGGGAAAGCACCGAAACTCTTGATCTACTATACGAGCAAACTGCATAGTGGAGTACCTAGCCGCTTTTCAGGCTCTGGCAGTGGTGCGGAATTTACGCTGACCATTTCAAGCCTGCAACCCGAAGATTTCGCGACTTACTTCTGCTTACAGGGGAAGATGCTTCCGTGGACCTTTGGCCAGGGGACTAAACTGGAGATCAAGCGTGGAGGTGGTGGATCCGGCGGTGGTGGCAGCGGCGGCGGTGGTTCGGGCGGCGGTGGCAGCGAAGTCCAGCTGGTCGAATCAGGCGGTGGTTCGGTTCAACCAGGCGGCTCTTTACGCCTCTCGTGTGCCTTTTCCGGGTTCAGTCTGAGCACGTCGGGAATGGGTGTTGGGTGGATTCGCCAAGCTCCGGGTAAAGGCTTGGAATGGGTGAGCCACATTTGGTGGGATGGAGATGAGAGCTATAACCCGTCCCTTAAAGGGCGGTTTACCATCTCGAAAGACACCAGCCGCAATACCGTGTATCTGCAGATGAACAGTCTGCGTGCTGAAGATACAGCCTCGTACTTTTGCGCGCGTAATCGCTATGATCCGCCTTGGTTCGTAGACTGGGGTCAAGGCACTACGGTCACCGTTAGCTCT DLX2289 SEQ ID No: 16EIVMTQSPSTLSASVGDRVIITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCLQGKMLPWTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSLSTSGMGVGWVRQAPGKGLEWVSHIWWDGDESYNPSLKSRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNRYDPPWFVDWGQGTLVTVSS DLX2260 SEQ ID No: 17EIVMTQSPSTLSASVGDRVIITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCLQGKMLPWTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTSGMGVGWVRQAPGKGLEWVGHIWWDGDESYNPSLKSRFTISRDTSKNTVYLQMNSLRAEDTAVYYCARNRYDPPWFVDWGQGTLVTVSS DLX2296 SEQ ID No: 18DIQMTQSPSTLSASVGDRVTITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPEDFATYFCLQGKMLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAFSGFSLSTSGMGVGWIRQAPGKGLEWVSHIWWDGDESYNPSLKGRFTISKDTSRNTVYLQMNSLRAEDTAVYFCARNRYDPPWFVDWGQGTLVTVSS DLX2295 SEQ ID No: 19DIQMTQSPSTLSASVGDRVTITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPEDFATYFCLQGKMLPWTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCAFSGFSLSTSGMGVGWIRQAPGKGLEWVSHIWWDGDESYNPSLKGRFTISKDTSRNTVYLQMNSLRAEDTASYFCARNRYDPPWFVDWGQGTTVTVSS DLX2332 SEQ ID No: 20EIVMTQSPSTLSASVGDRVIITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPEDFATYFCLQGKMLPWTFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGSVQPGGSLRLSCAASGFSLSTSGMGVGWRQAPGKGLEWVGHIWWDGDESYNPSLKSRFTISRDTSKNTVYLQMNSLRAEDTASYFCARNRYDPPWFVDWGQGTTVTVSS VH CDR2 of DLX2295SEQ ID No: 21 HIWWDGDESYNPSLKG humanized antibody VL SEQ ID No: 22DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWYQQKPGKAVKLLIYYTSKLHSGVPSRFSGSGSGTDYTLTISSLQQEDFATYFCLQGKMLPWTFGQ GTKLEIKhumanized antibody VH SEQ ID No: 23QVQLQESGPGLVKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDGDESYNPSLKSRLTISKDTSKNQVSLKITSVTAADTAVYFCARN RYDPPWFVDWGQGTLVTVSSVL DLX2332 SEQ ID No: 24EIVMTQSPSTLSASVGDRVIITCRASQDISNYLSWYQQKPGKAPKLLIYYTSKLHSGVPSRFSGSGSGAEFTLTISSLQPEDFATYFCLQGKMLPWTFGQ GTKLTVLG

While there are shown and described presently preferred embodiments ofthe invention, it is to be understood that the invention is not limitedthereto but may be otherwise variously embodied and practiced within thescope of the following claims. Since numerous modifications andalternative embodiments of the present invention will be readilyapparent to those skilled in the art, this description is to beconstrued as illustrative only and is for the purpose of teaching thoseskilled in the art the best mode for carrying out the present invention.Accordingly, all suitable modifications and equivalents may beconsidered to fall within the scope of the following claims.

1. An isolated nucleic acid molecule encoding a humanized antibodyagainst IL-1 beta comprising a variable light chain sequence having atleast 90% identity to SEQ ID No: 8; and a variable heavy chain sequencehaving at least 90% identity to SEQ ID No:
 12. 2. The molecule of claim1, comprising a variable heavy chain sequence (VH) as set forth in SEQID Nos. 9, 10, 11, 12 or
 13. 3. The molecule of claim 1, comprising avariable light chain (VL) sequence as set forth in SEQ ID Nos. 7, 8 or24.
 4. The molecule of claim 1, wherein the antibody has the sequence asset forth in SEQ ID No.:
 19. 5. The molecule of claim 1, wherein theantibody is an antibody fragment selected from the group consisting of aFab, a Fab′, a F(ab)′2, a scFv, a Fv fragment, a nanobody, a VHH or aminimal recognition unit.
 6. The molecule of claim 1, wherein theantibody is a scFv fragment.
 7. The isolated nucleic acid molecule ofclaim 1, wherein the humanized antibody comprises variable heavy chainCDR-H1, CDR-H2 and CDR-H3 sequences as set forth in SEQ ID Nos: 4, 21,and 6, respectively, or variants thereof, and wherein a) position 24 ofthe heavy chain is an alanine residue (A); b) position 25 of the heavychain is a phenylalanine residue (F); c) position 44 of the heavy chainis an isoleucine residue (I); d) position 56 of the heavy chain is aserine residue (S); e) position 82 of the heavy chain is a lysineresidue (K); f) position 86 of the heavy chain is an arginine residue(R); and/or g) position 105 of the heavy chain is a phenylalanineresidue (F); according to AH0 numbering; and/or comprising variablelight chain CDR-L1, CDR-L2 and CDR-L3 sequences as set forth in SEQ IDNos: 1, 2; and 3, respectively, or variants thereof, and wherein h)position 1 of the light chain is an aspartic acid residue (D); i)position 3 of the light chain is a glulamine residue (Q); j) position 20of the light chain is a threonine residue (T); k) position 99 of thelight chain is a glutamic acid residue (E); l) position 105 of the lightchain is a phenylalanine residue (F); m) position 146 of the light chainis a glutamic residue (E); n) position 147 of the light chain is anisoleucine residue (I); o) position 148 of the light chain is a lysineresidue (K); and/or p) position 149 of the light chain is an arginineresidue (R), according to AHo numbering.
 8. The molecule of claim 7,wherein the variable heavy chain further comprises at least one of thefollowing residues: (i) Serine (S) at heavy chain amino acid position 12(according to AHo numbering); (ii) Serine (S) or Threonine (T) at heavychain amino acid position 103 (according to AHo numbering); and/or (iii)Serine (S) or Threonine (T) at heavy chain amino acid position 144(according to AHo numbering).
 9. The molecule of claim 7, wherein thevariable light chain sequence comprises the sequence as set forth in SEQID No.: 8 and the variable heavy chain sequence comprises the sequenceas set forth in SEQ ID No.:
 12. 10. The molecule of claim 9, wherein theantibody further comprises a linker sequence set forth in SEQ ID No: 14.11. The molecule of claim 7, wherein the antibody has the sequence asset forth in SEQ IDNo.:
 19. 12. The molecule of claim 7, wherein theantibody is an antibody fragment selected from the group consisting of aFab, a Fab′, a F(ab)′2, a scFv, a Fv fragment, a nanobody, a VHH or aminimal recognition unit.
 13. The molecule of claim 7, wherein theantibody is a scFv fragment.
 14. The molecule of claim 7, wherein theantibody is a full-length immunoglobulin. 15.-20. (canceled)
 21. Avector comprising a nucleic acid molecule encoding a humanized antibodyagainst IL-1 beta comprising a variable light chain sequence having atleast 90% identity to SEQ ID No:8; and a variable heavy chain sequencehaving at least 90% identity to SEQ ID No:
 12. 22. The vector of claim21, wherein the antibody comprises a variable heavy chain sequence (VH)as set forth in SEQ ID Nos. 9, 10, 11, 12 or
 13. 23. The vector of claim21, wherein the antibody comprises a variable light chain (VL) sequenceas set forth in SEQ ID Nos. 7, 8 or
 24. 24. The vector of claim 21,wherein the antibody has the sequence as set forth in SEQ ID No.: 19.25.-40. (canceled)
 41. A host cell comprising a heterologous nucleicacid molecule encoding a humanized antibody against IL-I beta comprisinga variable light chain sequence having at least 90% identity to SEQ IDNo: 8; and a variable heavy chain sequence having at least 90% identityto SEQ ID No:
 12. 42. The cell of claim 41, wherein the humanizedantibody comprises a variable heavy chain sequence (VH) as set forth inSEQ ID Nos. 9, 10, 11, 12 or 13, and a variable light chain (VL)sequence as set forth in SEQ ID Nos. 7, 8, or
 24. 43-61. (canceled)